Estructura

 de  una  propuesta  de  proyecto  de  investigación  

 
Ejemplos  de  Introducción  
Se  presentan  tres  ejemplos  de  lo  que  se  consideraría  como  una  buena  introducción.  
Aunque  se  trata  de  artículos  y  no  propuestas  de  proyectos,  la  estructura  en  este  caso  es  
muy  similar.    
 
En  el  caso  de  la  detección  de  ondas  de  bajas  frecuencias  en  el  intestino  delgado,  el  texto  
inicia  con  una  descripción  de  la  fisiología  del  intestino  y  la  importancia  de  la  información  
proveniente  de  las  ondas  registradas.  Posteriormente  se  mencionan  algunos  estudios  
hechos  por  otros  investigadores  y  se  menciona  el  enfoque  que  se  seguirá  en  el  estudio  
en  particular  de  los  autores.  
 
En  el  artículo  de  cuantificación  de  daño  al  miocardio,  inicia  con  una  mención  a  distintas  
técnicas  que  se  han  empleado  en  el  pasado  para  identificar  y  revascularizar  al  
miocardio.  Menciona  la  importancia  de  la  enfermedad  y  algunas  cuestiones  sobre  
estudios  de  la  remodelización  del  miocardio  para  posteriormente  mencionar  las  técnicas  
que  han  desarrollado.  
 
En  el  caso  de  la  imagenología  por  tensores  de  difusión  o  tractografía,  la  introducción  va  
desde  lo  muy  general  (tamaño  del  cerebro)  y  técnicas  histológicas,  a  lo  particular,  
mientras  se  mencionan  las  ventajas  de  la  tractografía  sobre  distintas  técnicas  que  
generan  imágenes  en  IRM:  Mediciones  de  T1,  T2  y  de  densidad  de  protones.  
fía.  
  
  
Ejemplos  de  antecedentes  
En  el  primer  artículo  mostrado  abajo,  la  sección  de  la  introducción  es  muy  parecida  a  lo  
que  se  esperaría  en  una  sección  de  estado  del  arte  en  un  protocolo  de  proyecto.  Se  hace  
una  revisión  minuciosa  de  las  técnicas  de  segmentación  empleadas  hasta  ahora.  Sin  
embargo  en  el  protocolo  es  necesario  profundizar  más  o  explicar  con  mas  detalle  las  
técnicas  que  se  mencionan  en  las  referencias  bibliográficas.  
 
El  segundo  artículo  es  un  buen  ejemplo  de  introducción  (el  primer  párrafo  del  artículo)  y  
una  sección  que  podría  considerarse  como  antecedentes  (A.  Localizing  synchronous  
activity:  state  of  the  art)  incluyendo  un  estado  del  arte.  Sin  embargo,  en  el  caso  del  un  
protocolo,  la  explicación  del  estado  del  arte  debe  ser  un  poco  mas  profunda.  
  
 
 
Ejemplo  detallado  de  objetivos:  
El  documento  anexo  abajo  parrishesplan.pdf  es  un  extracto  que  la  National  Science  
Foundation  pone  a  disposición  como  ejemplo  de  cómo  llenar  los  formatos  para  las  
propuestas  d  e  proyectos.    
En  este  caso  el  primer  párrafo:  Capsid  structures…muestra  la  importancia  del  proyecto  
propuesto;  el  segundo,  A  model…  muestra  el  estado  del  arte  del  conocimiento  en  este  
campo.  
A  continuación,  se  presentan  los  objetivos,  que  en  inglés  se  llaman  “aims”:  
Aim1  To  define  structural  variation…  y  esto  viene  junto  con  la  hipótesis  específica  para  
este  objetivo.  Luego  se  muestran  los  objetivos  específicos  de  este  primer  objetivo:  a)  
further  define  th  estructural  flexibility…;  b)  determine  the  functions…;  c)  compare  the  
functions  of  capsid…  
Luego  se  plantean  los  otros  dos  objetivos  generales,  sus  hipótesis  y  sus  objetivos  
específicos.  
A  continuación  se  presenta  la  importancia  (significance)  de  estos  objetivos:  A1  entender  
los  procesos  de  infección  básicos;  A2  Barreras  críticas  para  tener  éxito  en  vacunaciones  
y  A3,  mejora  en  el  conocimiento  científico  fundamental.  
 
A  estas  secciones  siguen  las  secciones  de  innovación  (podría  ser  una  mejora  o  una  
aportación)  y  el  enfoque  o  “approach”  que  se  refiere  a  como  están  relacionados  los  
objetivos  entre  ellos  para  cumplir  con  un  objetivo  fundamental.  
 
 
Principal Investigator/Program Director (Last, first, middle): Parrish, Colin, R.
Relevancia del Proyecto
Capsid structures, variation and flexibility. This project brings together the skills of
laboratories at Cornell University and at Pennsylvania State University Medical Center to
provide a detailed understanding of the roles of structural variation and flexibility in the
parvoviral capsid, and their effects on receptor and antibody binding and the controls of
cell infection and host range. These are fundamental problems that apply to all animal
viruses, where the capsid must protect the genome in the environment, interact with host
molecules including cell receptors and antibodies, and undergo a series of regulated
structural transitions during cell entry to eventually release the genome for replication.
Viral capsid binding to host receptors and antibodies can have varying and often
unpredictable effects on infection, and those interactions also control many other
replication steps. Where these virus-host interactions are specific they can control the
viral host ranges.

Antecedentes
A model for understanding virus cell infection and host range control through
differential receptor binding. We study two viruses that differ in host range due to 3 or
4 capsid protein mutations that control specific receptor binding. Canine parvovirus
(CPV) arose around 1976 as a variant of feline panleukopenia virus (FPV), and caused a
pandemic of disease during 1978 and 1979. That virus has continued to circulate
worldwide as a serious canine pathogen, and has also evolved new antigenic, receptor
binding, and host range variants. FPV and CPV both can bind the feline transferrin
receptor 1 (TfR) to infect cat cells, and CPV gained the host range for dogs by gaining
the ability to bind the canine TfR. This new binding property was associated with
increased flexibility of 2 or 3 of the surface loops in the capsid that allowed CPV to
accommodate a glycan on the canine TfR binding domain. Flexibility in the capsid is
controlled by variation in hydrogen bonds, by cleavages of the VP2, and by ion binding.
Later steps in infection also involve changes in the capsid structure that release the viral
DNA or protein domains of VP2 and VP1. These viruses are targeted by antibodies that
can differ in their binding sites and in their ability to neutralize the virus. We will examine
a set of antibodies that detect specific capsid structures, examining the effects of
antibodies on TfR binding, and seeking to understand the mechanisms of neutralization.

Objetivo e hipótesis 1. Tiene 3 objetivos específicos

Aim 1. To define the structural variation in parvovirus capsids, and to determine
the effects on capsid functions and DNA release.
Hypothesis: That the capsids of parvoviruses undergo structural variation that is
important for infection. That occurs through the binding or release of divalent ions, by
site-specific proteolysis, or by variation in specific intra- or inter-chain bonds.
a) Further define the structural flexibility in the capsid through analysis of the structures
and to identify sources of variation using specific peptide and protease analysis.
b) Determine the functions of specific capsid structures by preparing mutants with
altered inter-chain bonds, divalent ion binding sites, or protease cleavage sites.
c)Compare the functions of capsid structures in mutant or naturally variant viruses to
reveal the structures and interactions that are critical for capsid stability, TfR binding,
and the processes of cell infection.
Aim 2. To define the structural interactions between various parvovirus capsids
and variants of the transferrin receptor or artificial receptors. Hypothesis: That
specific binding of capsids to the feline or canine TfRs is required for successful cell
infection, and those interactions are controlled by viral structures varying in structure and
flexibility.
1. a) Determine the interactions of the feline and canine TfRs with different
parvovirus capsids, examining cryoEM structures of receptor-capsid complexes
at moderately high resolution. By correlating residues on the capsid and TfR that
affect binding, identify the interacting structures.
2. b) Identify functional sites on the capsids by selecting for mutants of CPV or FPV
by growth on TfRs with mutant binding domains, or on receptors with artificial
binding ligands.
3. c) Prepare capsids with insertions that bind alternative cell receptors, and test
for cell infection.
4. d) Examine how flexibility of capsid loops controls interactions with different host
TfR - in particular receptors
with additional glycans within the attachment face of the receptor.
Aim 3. Use antibodies to probe the capsid structure, and also to determine how
binding to overlapping sites leads to variable neutralization. Hypothesis: That
antibodies can be used to detect variant structures in the viral capsid, and that the
specific position and orientation of binding controls the likelihood of competition with the
TfR, and neutralization of infection.
1. a) Examine antibodies with known capsid binding sites for their effects on TfR
binding, including the effects of cleavage with proteinases or after other
asymmetrically or symmetrically displayed modifications.
 2. b) Determine the effects on viral functions of antibody variants engineered with
increased binding affinities. Identify sites on the virus that do not bind antibodies
but that bind TfR, for example those subunits with cleavages in surface loops.
Specific Aims Page 31
Principal Investigator/Program Director (Last, first, middle): Parrish, Colin, R.
Relevancia
A) SIGNIFICANCE.
Información previa
A1) Importance: understanding fundamental aspects of virus infection processes
controlled by receptor or antibody binding, and the controls of viral host ranges.
The parvoviruses include many different human and animal pathogens, including the
long-known B19 virus which causes the childhood fifth disease and more severe
diseases of adults, as well as the recently identified human bocavirus and Parv4. The
adeno-associated viruses (AAVs) are parvoviruses that are not associated with disease,
but are being developed as human gene therapy vectors and the same issues of
receptor and antibody recognition are important for vector optimization. The viruses we
are studying in this model are the canine parvovirus (CPV) and its close relative feline
panleukopenia virus (FPV), which bind to the host transferrin receptor type-1 (TfR) to
infect cells (53). The parvoviruses have a 25 nm diameter T=1 capsid that is assembled
from 60 copies of two or three versions of a single capsid protein, and the single
stranded DNA of the virus is packaged into the pre-formed capsid by the action of the
larger non-structural protein (NS1). Although those capsids are remarkably robust and
survive in the environment, structural variation results in viruses with different properties,
and those also show structural changes during the process of cell entry and nuclear
trafficking. The simple and well defined structures of the parvovirus capsids, the known
properties of the TfR, and the well characterized antibodies available for these studies
allow us to examine several processes of viral infection.
Liga la investigación a la salud pública
Variant viruses with extended host ranges can cause new outbreaks or epidemics of
infectious disease. The viruses that we are examining include the comparison of such a
system, where one variant arose as a pandemic pathogen in a new host through the
acquisition of mutations in the capsid protein that altered its structure to change host-
specific receptor binding, and also to change its antigenic structure.
Problemas y barreras en el campo
A2) Critical barriers: to antiviral therapy and vaccination success. Animal viruses
are complex biological machines that engage host cell receptors and undergo a series of
varying structural and functional changes to allow cell penetration and release of the
genome for replication. Those infection processes are key to the success of any virus,
and are targets of various anti-viral drugs. A better understanding of the details of the
general processes involved will likely allow the development of more effective and
broadly acting antiviral drugs. Although antibodies are critical components of immune
responses of all vertebrates, in many cases they are poorly effective so that viruses
maintain persistent infections or vaccines do not work well. Understanding the
underlying rules that determine how antibodies bind to viruses and block the processes
of cell infection will reveal how effective antibody responses might be elicited against
different viruses.
Potencial para lograr avances en el campo
A3) Improvement of scientific knowledge: understanding fundamental viral
mechanisms and clarifying textbook knowledge of virus structures and functions.
This project addresses several mechanisms important for all viruses of humans and
other animals. In general terms those include understanding viral recognition of cell
receptors, how changes in receptor binding sites lead to alterations of binding and host
range, capsid and receptor structures and the interactions that control uptake and
trafficking within cells. During each of these steps the viral proteins must assume the
correct conformations, bind receptors with the correct contacts, and in the process
undergo a variety of structural transitions to release internal peptides, protein domains,
and the viral DNA. Here we will investigate the roles of flexible and variable structures in
the parvovirus capsid and show how receptor and antibody binding control cell infection.
B) INNOVATION.
This is an unusually complete model for understanding virus structures and functions
involved in cell infection and host range control, as there are few other viruses where the
ancestors and descendents of a host- switching virus that caused a pandemic of disease
in its new host are available for analysis. The laboratories presenting this proposal are
the only ones working with this model in any detail, but we have been able to explain
many aspects of the process, from the evolutionary processes allowing emergence, to
the identification of specific receptor binding as a key step that lead to the extended host
range of the virus [reviewed (52)]. There are several fundamental issues being
investigated in these studies, including being able to obtain a detailed understanding of
the variation and dynamic properties of viral proteins, how those structural changes
control receptor attachment and cell infection, and that analysis is complemented by
studies of antibody binding and its effects on receptor binding and infection. By analysis
of naturally variant or mutant viruses, receptors, and antibodies we will gain a better
understanding of the functional properties of the capsid-ligand structures and alterations
in binding properties. We use a variety of advanced methods and structural, biophysical,
biochemical and functional assays in these studies. While many of the methods we
propose are being used in viral studies (including by our laboratory), the proposed
studies include combinations of those methods that are being used in novel ways to gain
a complete understating of the mechanisms involved.
Research Strategy Page 32
Principal Investigator/Program Director (Last, first, middle): Parrish, Colin, R.
C) APPROACH.
The three related and overlapping specific aims are directed at understanding: (1) the
roles of structural variation and flexibility in controlling capsid functions; (2) the
interactions of viral capsid structures with different receptors, and the mechanisms that
lead to infection; and (3) the antibody binding to capsids and resulting competition with
receptors and mechanisms of neutralization
C1) Aim 1. To define the structural variation in parvovirus capsids, and to
determine the effects on capsid functions and DNA release. Here we seek to further
define the structural variation of the capsid, including sources of asymmetry. In these
studies (and also in those of Aims 2 and 3) we also define the effects of those changes
on the functions of the virus, in particular its interactions with receptors or antibodies.
Muestra resultados previos o preliminares
C1a) Background – brief summary of literature and preliminary results.
C1a1) Functional variation and flexibility of parvoviral protein structures. The
structures of parvovirus
capsids vary between different viruses, and in our previous work we have shown that
small changes control significant variation in the viral properties including receptor
binding, host range, and antigenicity [some recent examples are: (19, 21, 22, 24, 26, 31,
45, 46, 49)]. Among the functional flexible or variable structures seen are loops with
varying numbers of intra-chain and inter-chain bonds, loops which bind or release 2 or 3
divalent ions, the possible formation or disruption of disulfide bonds, protease cleavage
of a proportion of the proteins, or opening and closing of cylindrical pores at the fivefold
axes which allow VP2 N-termini and the viral DNA 5’- termini to pass to the outside of
the capsid (14, 15).
In the studies proposed, we are using information derived from many different
parvoviruses and adeno- associated virus studies, and while space does not allow a
review of that literature, those include studies of structural variability and flexibility in the
capsids that occur under various conditions, including at low pH, ion removal, after host
cell, receptor, or antibody selection, ubiquitin modification, or after binding to
sphingolipids or receptors [a small number of the relevant references are: (13, 16, 30,
34, 36, 38, 41, 47, 48, 56, 61, 65, 66)].
Explicación de la biología para los revisores del proyecto
C1a2) The roles of structural variability and flexibility in viral proteins. Functional
variation in structure is a key property of many virion proteins, with well recognized
triggers for conformational change including low pH, ion binding or release, receptor
binding, or protease cleavage, although other more subtle and less well understood
changes are also likely to be common. The changes resulting often allow membrane
penetration and/or fusion, or the complete or partial disassembly of the virion to expose
internal components and to allow viral trafficking and genome release for replication. As
examples, structural changes in the gp120 of HIV are induced upon CD4 binding,
allowing binding of CCR5 or CXCR4 co-receptors that mediate infection (59). The
binding of receptors to the picornavirus capsid can result in structural changes (7, 55),
and antibodies binding to the flavivirus E glycoprotein can trap intermediate
conformational forms of the protein (29, 39). In addition, variation in viral protein
structures can result in antibodies binding to forms that differ from those seen by the
receptors, allowing evasion of at least part of the antibody response [e.g. (10)].
Submolar cleavages, asymmetric structures such as portals, or incorporation of minor
proteins into virions may be common in viral proteins but are often hard to define in
detail and therefore not well understood. Explica las metas Here we seek to define the
functional connections between the variation in the parvovirus structure, including
submolar variation, and its functional significance for virus receptor and antibody binding
and the infectious cycle.
C1a3) Asymmetry of functional sites in viral structures. Some of the flexible
structures in parvoviral capsids are asymmetric, being present in only a small proportion
of the capsid subunits. Examples include those resulting from cleavage of surface or
internal loops of a small number of the VP1 and VP2, the inclusion of 5-6 VP1 molecules
per capsid, or the packaging of one ssDNA molecule with its 5’-end protruding from the
capsid (13, 15). These types of asymmetrically variant structures are likely present in the
capsids of many different types of viruses, but can be difficult to detect without specific
probes, and would not be seen after X- ray or cryoEM analysis of viral capsids that
involve symmetry averaging for the reconstructions.
The VP2 within the capsid can be cleaved at several positions. The cleavage of VP2 to
VP3 occurs only in full capsids, and occurs between residues 15 and 24 depending on
the specificity of the proteinase, and must involve exchange of cleaved and uncleaved
N-termini through the fivefold axis pores of the capsid (15, 45). That VP2 to VP3
digestion is temperature dependent, most likely because the pores (and perhaps other
parts of the capsid) have to expand to allow exchange of N-termini (15, 45). Divalent
ions bound within the capsid are associated with changes in the flexibility of several
surface loops, including those involved in binding to sialic acids (60).
Research Strategy Page 33